title: Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).
abstract: Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1. With this simplification, we found that elimination of the lysine pairs Lys-253/Lys-256 and Lys-270/Lys-289 resulted in increases in Kd of 1240- and 100,000-fold, respectively. Each pair contributed additively to overall affinity, with 61% from Lys-270/Lys-289 and 39% from Lys-253/Lys-256. Furthermore, the Lys-270/Lys-289 pair alone could bind different single CR domains with similar affinity. Within the pairs, binding contributions of Lys-270 ≫ Lys-256 > Lys-253 ∼ Lys-289 were deduced. Importantly, however, Lys-289 could significantly compensate for the loss of Lys-270, thus explaining how previous studies have underestimated the importance of Lys-270. Calorimetry showed that favorable enthalpy, from Lys-256 and Lys-270, overwhelmingly drives binding, offset by unfavorable entropy. Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a CR domain, with two such pairs of interactions (requiring two CR domains), appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
authors.forename: Klavs
authors.initials: K
authors.lastname: Dolmer
authors.forename: Andres
authors.initials: A
authors.lastname: Campos
authors.forename: Peter G W
authors.initials: PG
authors.lastname: Gettins
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D002942:Circular Dichroism; D005453:Fluorescence; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D050505:Mutant Proteins; D008239:Lysine
keywords: Fluorescence; Isothermal Titration Calorimetry; Ligand Binding; Lipoprotein Receptor; Lipoprotein-like Receptor (LRP); Low Density Lipoprotein (LDL); Lysine; Receptor Structure-Function; Receptor-associated Protein; Thermodynamics